Blokland, E.

Breur-Vriesendorp, B.S.

Dekker-Saeys, B.J.

Goulmy, E.A.J.M.

Ivanyi, P.

Linden, S. van der

Pool, J.

Post, F.A.

Waal, L.P. de

Leiden University Scholary Publications

3 , , ' . ,iRwll ,ü s \ l e \ is ! ι missnif,c ι >| n s willIne 655Blood Lymphocytes from AnkylosingSpondylitis Patients Fail to InduceDisease-Specific Cytotoxic Τ Lymphocyteslshipsaaon Birg i t ta S. Breur-Vriesendorp, F r a n k A. Post,Leo P. de Waal, Eis Blokland, Jos Pool,3 m a Sjef van der Linden, Betty J. Dekker-Saeys, Eis Goulmy,and Pavol Ivanyi1dABSTRACT The intnguing observatton made by Geczy et al (1} showing the possibüity of generatingspectfic ankylosing spondylitis—cytotoxic Τ lymphocytes by presentmg HLA-B27 AS¥ cells asantigen-speafic stimulator cells prompted us (by ustng Geczy s approach) to identify cytotoxic Ίlymphocytes specific for this apparent B27+AS+ target structure Penpheral blood mononuclearcells (PBMC) of 21 healthy B27* indtviduals were stimulated in pnmary and in short-termcultures wtth PBMC of an HLA identtcal sibling suffenng from definite AS (n = 12) Inaddition PBMC in vitro modtfiedby Geczy bactenalproducts from two healthy B27+ individuals were used to sttmulate B27*AS lymphocytes (either autologous or from α healthy HLAidentical sibling) Effector cells raised in pnmary AS versus AS+ and AS versus modifiedB27 mixed lymphocyte culture combmations showed no prolifetative nor cytotoxic activity at allThe \canely observed cytotoxic reactivity of restimulated mixed lymphocyte culture was not restncted to AS B27+ cells These results demonstrate that PBMC from ankylosing spondylitispatients fall to induce disease specißc cytotoxic Τ lymphocytes and suggest that an ankylosingspondylitts—related modifiedB27 structure does not extst at hast in thepatient matenal testedABBREVIATIONSAS ankylosing spondjhtisCML cell mediatedl xurpta lymphocytotoxicityCTL cytotoxic Τ lvmphocyteID limmng dilutionm i i s Ntv. minor Η m i n o r histocompatibilityMLCMLRPBLPBMCmixed lymphocyte culturemixed lymphocyte reactionpenpheral bloodlymphocytespenpheral bloodmononuclear cells/ rom the Central Laborator) of the Netherlands Red C ross Blood Transfu ιοη Sen ice and Laboratoi y fortop\ inf, of Ϊ xpertmental and Clintcal Immunology Vntversity of Amsterdam Amsterdam (B S ß V ΓΑΡ L Ρ WIS £,ivcn on Ρ I} the Department of Immunobematology Vniverstty Hospttal Leiden (Ε Β J Ρ L G ) the DepartmentAyL of eich ofRheumatology Umierstty Hospital Maastricht (S L ) and the Jan tan Breemen Institute for Rheumata!the author ogy Amsterdam (B J D $ } The Netherlandsuchor Ih i s Address repnnt requests to Dr Ρ hanyi clo Publtcation Secretanat Central Laboratory of the Netherrtismg ami lands Red Cros\ Blood Transfusion Service PO Box 9406 J 006 AK Amsterdam The NetherlandsRecened May 31 1988 rensed December 22 1988es •?() ϊ astHuman Immunoiogy 25 149—155 (1989) 149© American Society for Histotompacibility ̂ nd immunoßenetics 1989 0198 8859/89/33 50nircd Starts150 Β S Breur Vncsendorp tt ilINTRODUCTIONThe association of HLA B27 with ankylosing spondyhns (AS) still is one of thebesr exampies of the association between human leukocyte antigens (HLA) anddiscase Either thc B27 gcne product ttself or a discase susceptibihty gcne prod1 uct in tight linkagc disequihbrmm with B27 must account for the observedassociation betwccn HLA and diseast So far no evidente favonng onc of thclattcr possibilitits has been prescntedIt has bcen reported that antibactenal sera can distinguish lymphocytcs ofB27 AS paticnts from B27 hcalthy blood donors [2—5] These hndings suggcsted an interaction of enterte orgamsrns (or their produets) «irh B27 Indcpcndent laboratoncs have never sueeeeded in rcproducing these rcsults [6-8] although blind Workshops confirmtd fhat celäs from AS paticnts could bcdistinguishcd from control cclls wtth thc Gcczy scra [9-11]Tbc Jatttr observanon led to thc hypothesis that therc might exist an ahen ormodified strueture on AS B27 cells If so thc qucstion could bc riistd whetheraliogenuc Τ cclls also rccognizc a B27 associated strueture on lymphocytcs of ASpaticnts This possibihty was exammed by Gcczy et al [1] who reported thcrecogninon of an AS associated strueture by cytotoxic Τ lymphocytcs (C1L) In[wo independent laboratoncs we performed sirmiar expenments wich an identical genctic design In our hands no detcctablc prolifcrative or CTL responseswerc induetd in mixed iymphocytc eultuics (M.LC ) of teils from hcalrhy individuals snmuiatcd with ceils from HLA identical Ab paticntsMA TFR1ALS A N D MLTHODSCelli Penphcritl blood lymphocytcs (PBL) were isolitcd frorn dciibnnatcdblood of randomly sclectcd hcalrhy B27 blood donors of dehnitc AS j^dticnts(aecording to thc New York entena) and of hcalthy B27 sibhngs who wereHLA jdentjeal with the AS proband but did not have evidcncc of sactoihittsThis radiologic sign can bc considered as a condmon sine qua non for thc dia^nosis of AS Thc cclls were used fresh or were stored in liquid nitro^en until useModified B27 AS eclis were obtained by mcubatinj, cclls with tht. euiturefiitrate K4^ (obtained from Α Γ Geczy) aecording to thc protoeol desenbed inMaterials and Mcthods by Gcczy et al [1 ] C ontrol teils were incubated with theeuiture hl träte 199MIC Thc prolifcrative ac ivity of efrector cclls wis measured in MLC by m< ubating 5 X 1 0 responder ecils with 5 x 1 0 irntliarcd stimuLitor cclls m 200/xlof eulture medium in mit,runter platcs After 3 days of Intubation Η thymidinewas added ιηά 24 hr larer rht jncorporation wis measuredCJL The t;encration ot AS speeihe effector eclis and thc tcctmg for specifiteytotoxic activity using the stand ml ccll mediatcd lympho>.ytotoxicity (CML)assay have becn carncd out atcordin^, to the protoeol desenbed in Materials -indMcthods by Geezy et a! [l]Cll hnes In a limitm^, dilutton (LD) eulture systern CTL Jines werc ^encratedGradcd numbers of responder PBL (lO 5 -^ x ΗΓ) were stimuUted with \{Ϋirradiatcd PBL frorn an HLA tdtnticai AS dffecied sibhng m 200 μϊ of eulturt.medium containmg intcrieukin 2 (1L 2) (40 units/ml) After 6 days of nuubaiiontwo sample s of 50 μλ of thc ttlJ eulture of each wel! werc tested for spccifictytotoxtcity against stimulator and another AS B27 target ccll in ihc CMLAnkylosinj, Spondylitis in I I C dls 151assay The rcmamini; teils in individual wclls werc restimulited with K)"1 scimulacor ccils in 100 μλ fresh medium Another strcening for cytotoxiuty was performet! dt day 12 Ibosc ccil eulturcs that were found to bc positive on bothtarget eclis wtre trinsierrtd into welk tontaining i ml of culture medium andwtre rtstimulated with 10' stimuUtor teils at da>s 1 3 and 19 At days 15 22 and24 1 ml of medium containing II 2 wis added Ccl! cultures with abundantprohfcration were divuied Fhe CTL hnts were tested for AS speeihe B27 associated cytotoxieity at diy 25 a^ainst ehe stimulitor cclls and agiinst two additiondAS B27 and two AS B2"7 tarnet cdh at an effettor to target rano of 10 1ithe number of tirgtt cclls was Α x 101)RFSUI TSThccytotoxic ictiviti^s of etfector ctlls genciatui in ? i AS /AS HLA identKalsibling rcspondcr/stimulator piirs within 10 familici md in thrtc AS /AS HI Αclass 1 identit il (B27 subtypc-muchi.d) class l l-nonidcnucal unrclatcd combinations (ste Tiblcs 1 and 2) were testtti in the CML assay In onc ί ILA idcncicalsiblmg pair (both healthy B27 individuals) cHcctor teils were gentrated usingstimulitor ccils t h u wtre modiiicd with either a K h or Γ99 (control) culturesuptrnat nit f urthtr inorc cells from onc randomly selecttd heilthy B2"1 blooddonor v/cre sttmuiated with lutologous ecils either Κ β or Γ99 motlifted Cytotoxiiity was mc isured after 6 days ot eultunng as well is after rcsnmulation oiC fL on d ly I 4 o f t u l t u r e is destribcd m Tablcs I and 2In cornbin itions ot HLA identieil siblings (AS AS ) the MLC wis ilv, lysiound to bc net,itive (proliteration was oniy tested m the combinations fiomAmsterdam) In nonc of the HLA itlcntical AS /AS sibling combinations didthe CML lssiy show spccifie lysis above that of the medium control of thesttmulatoi or thiri! ρ irty AS Β }Ί tirgct teils C clls irom the samt respondersstimuhted agunst 1 ILA chss 1- anti tlass ί ΐ-mism itehed celis showed goodprolifer ition and elfcenvclv Ivsed (M)-60 r r ""'C r rele isc) stimulator target etllsNo speeiiie 1>SIS oi AS tirt,ct teils wis found in tht AS /AS combinations ofHLA tliss I - identitü unr t lucd individuals diffenng in tlass II antigens although all hui ι positive MLC Morcover in nonc of the CTL riiscd againststimul xtor cells modified with b icten il culture hlrr ites was AS sp^tiht cytotoxititj ilttettctl Reprcseni iti\e results of the expenments ire givcn in ligure 1I ckolf ind Shaw {12} d t s t r ib td in 1 D eukure s\stcm \s α pnmir> in vitromethod to >,entrite HLA rtstrieted CTL specific tor minor hutoeompatibiiitiiminor 11) intimens bc tween HLA identit il siblinys The possil iht; ot producint,AS spccilit ( II in such in Ι Ο System u is tx immc 1 I r o m i o i r A S /AS siblmgΓΑΒΙ1 I ( clls used in Amsterilim foi the induction oi AS speeitic C TL( nihin ! ι K L S ] )i I r L U I SB 2 7 AS(Μ Π( ι r m i y u M I I I M I I i l ( u ( h u I i n r t ι [ k r n I t r m i l i t π ί Γ μ ι t i . l l ί I I (I n s Ir ii f r \ s \ S s i l i f. ι i n ^ k i i ^ i i s I t s π ι t ί π ι Μ i c t n i l i in I A k t h > iK152 Β S Breur Vncscndorp et 1]TABLE 2 Cells ustd in Leiden for the inducnon of AS specific CTLCombinaiRtspondtr cclls Snmulator cells(r t ) (s t 1HLA class I - a n d class I] idcntind class II idcnhcal B27 AS(» 11B27 AS(» - 1)B27 AS AuLoloBmsdc r t IUnrdatcd B27 AS in - I)UnrclitcJ 1327 AS (» ')B27 AS ALitologoiLS li t r t )K43 miidificd Spccificsc K4 5modi(lcdSpccLhc s c F99 moiUficdSame s c Unrtlaitd B27 AS (» I)i)> modifitd Unrtlattd B27 AS (» 2)SptufLis t K 4 1 m ) l i l t lSpecific s t 1 9 ; modifitdUnrdatcd 1 3 " AS (/ 1Unrclatcl B27 AS « 'Auioli)C Γ CmuJifAutol"C Γ tmotdiK4iic IIΓ9;icllAll t-ir^cc cclK arc usctl wiihouc uiy irnto^cnic. stirnui^tion ίι L 6 <Jays PBL IÜLHI eil u CIIL pro to t )l usc I tayO c t / y t t li {I i) as WLÜ as stimulitL f f r ^2 hr v. th pi tyrol i tma^Jut i f imh The rcsponiicr ccll p o p u h n m v.is «.timulitt. 1 with ctUs from ehe I ILA ι lei tical Mblnif, <J )iu r m ) Uhc 1 witht ithcr Κ ιΐ (modifyini, f icior) or U 9 (t t ^ t i v i t incrol s u p c r r u t i m l both t u l t u r t h l tnces t i m i In m OHL ol rlicc r o s s r e ^ t v e organisms (SLC M a t c n a J w m l Mcchals Gccz) u dl i J JT h e resj )ii ler teil p o p u U n o n hai bt-tn mo I hc 1 wich K 4 ^ as v.tll is w rh I 9 J an 1 ι SLII as snmuUi >r/iari,ctteilspairs Τ ccll hncs wert gencrated and cested for AS sptcific cytotoxicity SomcCTL ltncs were cytocoxic j-Or stimulacor targtt ceils somc third party AS B2*7as well as AS B27 target cells Othtr Τ ctli lmti> iysed somc chird party tirgctcclls but were not rcactivt with the spetihe stimui itor cells Nonc ot the ccll lincswere ablc to disnnguish AS from AS tirgct ccllsDISCUSSIONOur expenments cirncei out in ι stmitar way to thosc ofGcczy et al [1] did notproducc the same rcsalts Duc to vanations between Jabonronts jr is difhcult toiisccrtain whetber the techniques employcd were rcaliy tdentical Howevi r wcare not aware of important tethnical differenees that might aecount for the observed discrepancics On ehe otlicr iiand ehe paticnt (and control) stl<^tion eouldplay a role In the work ofGcczy the positive rcsults emerged irom two HI Αidentica! AS /AS sibiing purs wc have examintd 21 such combinations (secTablcs 1 and 2) without iinding t smgic positive tombination Aithough rhcicmay bc some dificrenecs in the paticnti examincl it is difncult to aeeept such acnticai degrec of discrepancy in the elmical dugnosis of the diseasc In fact thelatter cxplanation for discrepancy can be exeluded due to the pre^ence of theseroiogically detcctable Geczy factor on the ceils of Durch AS patients inprevious blind Workshops [10 l l j Morcovcr in our expenments no CTL activlty WJS measured in the eultures with K43 modified stimulator cellsAnLylusing Spondylitis and Τ CciK£ 701S 6O£ 50Ι(Λ40302010- Q 2 A3A5 1 10 1 50 1effector target ratioI l G U R l l I l lustruivt Lw u t a n t r i t t I in i l ) 111 'tu il AS /AS , ur«.. i a t ior K l i -ni t die AS s u m u h t o r π ιm p k i of abstritt, ol AS spccilit e\kntKtl Ab /AS sibli^ pur^ (2){ (\) AS B-1"7 h l ) ) ! lom rs snn! t t l U AS SflttlllL LJtOtOXK l(.ti\li n l l s l l A Μ ^A) Alio«.p(.<JfK.e>•toxieity i ffef tor CLIISj i r t h r c d H I \ cl iss l -.ulue I with iutoloj,ousis tLsti. i an rhe reMC icn\tt\ otütti.ww CLSte! an rhtLLIN (111 2B UilThe frirneviork oi theuiri AS ( II lints in shorin some e iscs \\ is not rcstrre< of ι or i\ .txptnments his been cxie rni teil (tilturt s 1 lovu 'OVRIf) ixtWtLil J Π Α jd-icd to B27 AS rir^cti^ens I rom the resuitsin tone lüde th ir ill eraied ii m ippropn irc illointigen wis present·>r eeJls used werc ihit r» intlutc profjferanontie i b) efforr1; to protiueer no AS speufit C Tl hncsim ü -.ibhnt, ρ urs observedIts ind ιηιφΐ bc duc t*i the:ir ( ur eontrol expenments>n Fffeetive C FL coulti bt.tl for Stimulation All stimJ and tould be lecoeniztd154 Β S Brcur Vncscndorp u alby specifically raised tffector cells in HLA ciass I~ and class II-mismatchedcombinations However in our expcnmental assay the oiseast and/or B27associatcd strucrure apparently is not anngtnic cnough for AS spetifk CTLinducnonIn conclusion for reasons which are not clcar to us we have not becri abic toinduce AS specifk CTL by MLC of B27 AS and B27 + AS+ cells or by B27 AScells modified with K43 supernatant culturc fiitrate The possibic modiücattonof MHC anttgens on cells with the DR2/DQ1 haplotypc of paticnts with narcoicpsy has becn studied m a sjmilar geneticai set up by Strohmaier et al [13]Narcoltpsy is the diseasc with the strongest HLA dssociauon nearly 100% of thepanents arc DR2/DQ1 ·" It was reported that no disease related alttration ofDR2 and DQl molccules on cells of narcolcpsy patients couid bc deteeeed bynormal Τ iymphocytcsFunher expenmems may eiucidart wherber djscasc specific struetures do existon lymphocytes of paticnts with HLA associatcd discases and if so which ofthe variables are umquely cntical for the activation of disease specific Τ lymphoCytesA( KNOWI t DGMINTWe chank all patients and their farmly members who volunatred to pamapate in rhisstudy Professor Dr C Ρ Fn^elfnct is icknowledged für his cntical readin^, of theminuscnpt dtul Professor Dr J J van Rood for hclpful tistussion We th ink Mrs IOerntsen for secrcttnal issistanceTliis study was fmantially supporttd by the Dutth Kidney fouiulation (^rani noC87 69^1 by the Swiss National Research I und (μ-int no ΐ 912 0 Ηΐ) ind m p irt byL,rants from the Duah 1 oundation for Medieal and Health Research (MI DIGON) ind theJ Λ Cohen Institute for Rid^i itholoj,y ml R uiutiun Protettion (1RS)REi-LRENCES1 Gec^y ΛΓ McGui>,an Lr SullivinJS rdmondsjP Cytotoxic Γ lymphoc^tes aj,iinstdisease issomtcd letermm uu(s) in ink> U sinj, spon lylitis | I xp Mc 1 16I9WU862 Sea^er Κ Bashir HV Gee/y Ab Tdmonds J De Vere Iyndill Α Evidente ior ιspecific B27 associarcd ce) surface marktr on iymphocyfLs ut pintnts with ankylosinf, spondyhtis N u u r t i 7 7 i 8 1979λ Gcczy ΑΓ Alexander Κ Bashir HV Edmunds J Α faaor in Kicbsielli ulturefiltrates spccificdly modifies in HI Α B27~associated teil surfaLC component Naturc28^ 782 19804 OrbanP SullivanjS Gec/y AI Couhts Ν Bashir HV Α fictor sin d by lymphobhstoid ceil lines o( HLA B27-positivc patients with ankylosmj, soondylitis spccilicdlymodifics the cells o^ HLA B27-positive normil individuai·; Cltn ί κρ Immunol5^ 10 198Ϊ5 Sullivan JS Gec^y AI Ihe modiifcinon ot HLA B27~positivt lymphotytes by theeulture filtrates of Klebsicll ι Μ 1 li TS 1 is t rneubohe Uly active process Clin ΓχρImmun»! 62 67^ 19«^(i Strurlicis GR t ross ic ictivity if uni Klaisitlli KiS nitistrum md l>mplioi.yKsfrom HI Α B27-positive pdtients wich ankylosinj, spondyhtis Lancec ι 761 1W>7 Gcorfeopoulos Κ Carson ÜW GoodacreJA Pain RH Α reinvestigation of the crossreactivity between KIcbsiella and I ILA B2 / in tht aetiology of ankylosing spondyhtis Ciin Exp Immunol 62 662 1985wnj, Spomlyliti·, md I U l i s I5S8 Sjn^h Β Mil tonjD WoodrowJG Ankilosmj; spond>lim HLA ΒΓ md KlcbsicllaΑ study oi lymphocytc rc ietivit) of inu KicbsK.Ui scrt Ann Rheum Dib |5 19019869 M t G u i K i n l l G t w y A l Prunlcrsast JK U m o n d s J P Hir t H H Bashir HV HLAB27—assoti uct! truss rcacnvc mirkcr on tht cdls ot NLW Zcdiantl pacicncs wichankylosin;; spomly lins Ami RliLum Dis l1) 1 1 t 1 JH610 R o o J J J v i n v a n U t t i M u i A lvan>i Ρ ( HS Α Hrcur Vricsindorp BS D t k k t r S i t v sAJ Klihtra Α van KrL^icn ί fjiimi conlitmituin ai Gcc^y t-itfor in ankylosmi,spondylilis Limct n ' ) n I486I ! G u i ) AI v i t i l u u w i o A w n R i x i d l l lv in s i l> B n u r B b C US Α Blind lonlnmation m Leiden oi G t c / j f iLtt>r on ι he uc lls (il Du a h puicnts with ankylosin^, spon !jIltis l lum lmmunul Ρ '19 1 )S(i12 i L k o l l W A Slil» S Primirj i m ltrt> M m r ition n U vtt>tu\ic n l l s speeihe lor liummmumr lusttKomp itibilnj i n n r e m between HLA ldcntital sibhnjis J lmmuno!I1J 1 /•>(> 19X111 Strohmuci Ρ Mulk r I ckh irdt t . M L i i r l » L r t k Mulkr } i l h irdt l lnvislifiitionson tlic tcllulir dLtLttion iil ι possiblc i lared seil an η m o k p s ) ass )u «cd DR2/OQ1 haplotipis Iniimmobiolo,,) P i '61 I9h^

Blood lymphocytes from ankylosing spondilitis patients fail to induce disease-specific cytotoxic T lymphocytes.

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Blood lymphocytes from ankylosing spondilitis patients fail to induce disease-specific cytotoxic T lymphocytes.

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